Correlation Study Between Adipose Triglyceride Lipase And Hormone-sensitive Lipase Expression In Adipose Tissue And Insulin Resistance

Epidemiological studies show that there is an obvious increase of type 2 diabetes mellitus frequency worldwide. Insulin resistance (IR) is the most important pathogenesis for type 2 diabetes mellitus, which is closely correlated with obesity, hypertension, dyslipidemia, and so on. Insulin resistance is the condition in which normal amounts of insulin are not able to uptake and dispose of glucose in peripheral tissues. Genetic and environmental factors play major roles in the development of insulin resistance. Based on the recent data, insulin resistance is related with multiple pathophysiological processes, such as lipotoxicity, glucose toxicity, oxidative stress, mitochondrial dysfunction, and so on. Our previous study has demonstrated that free fatty acids (FFAs) play a pivotal role in the development of insulin resistance.FFAs in the circulation are mainly derived from diets and hydrolysis of triglyceride storage in adipose tissue. Triglyceride in adipose tissue is hydrolyzed into FFAs and glycerol under the effect of lipases, which is called fat mobilization and offer energy to the metabolism of organism. Hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) are the major enzymes in fat mobilization, which are responsible for more than 95% of the triglyceride hydrolase activity. The expression and activity of ATGL and HSL are closely related with FFAs level in the circulation and may play roles in insulin resistance.In the present study, we establish the insulin-resistant model of rats induced by high-fat feeding to detect the expression of ATGL and HSL at mRNA and protein level in adipose tissue; and give rosiglitazone for drug intervention to explore the effects of rosiglitazone on expression of ATGL and HSL. Meanwhile, we also investigate the expression of ATGL and HSL in subcutaneous and visceral adipose tissue of insulin-resistant people to explore the relationship between disordered fat mobilization, fat-depot specificity and insulin resistance, providing a new study direction of improving insulin resistance. This paper contains two parts as below:Part One: Expression of ATGL and HSL in adipose tissue of insulin-resistant rats induced by high-fat feeding and effect of rosiglizatone interventionObjective: To investigate the effects of high-fat diets and rosiglitazone on the expression of ATGL and HSL, key enzyme in the process of triglyceride hydrolysis, and to explore the role of disordered fat mobilization in insulin resistance.Methods: 40 male Wistar rats aged 4-5 months were randomly divided into normal control (NC) group (n=14) and high-fat diet (HF) group (n=26).The rats in control group were fed with a regular low fatty acids diet; while the rats in high-fat diet group received regular diets mixed with 30% lard, containing 59.8% fat, 20.1% protein and 20.1% carbohydrate as a percentage of total calories. After 4 weeks feeding, the blood sample was collected from angular artery for the biochemical analysis; insulin sensitivity was evaluated by glucose infusion rate (GIR) of hyperinsulinemic euglycemic clamp technique at the end of the fourth week (six rats in each group). After the evaluation of insulin sensitivity, rats were randomly divided into high fat diets group and high-fat + rosiglitazone group (RSG), and each group contains 10 rats. The rats in RSG group were given rosiglitazone (GlaxoSmithKline, UK) 3mg per kg body weight through intragastric administration once a day for 4 weeks; while the rats in other two groups were given equal physiological saline. At the end of eighth week, the rats were killed after hyperinsulinemic euglycemic clamp test and oral glucose tolerance test. Retroperitoneal adipose tissues were taken out freeze-clamped with copper clamps precooled in liquid nitrogen and were stored in -70℃refrigerator. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect mRNA expression of ATGL and HSL; while western blotting was used to detect protein expression of the two enzymes.Results: 1.At the end of fourth week, the glucose infusion rate was significantly lower in HF group than that in NC group. Insulin resistance was successfully induced. At the end of eighth week, compared with NC group,the levels of fasting blood glucose,triglyceride,cholesterol,FFAs,fasting insulin and relative visceral fat mass were significantly higher and glucose infusion rate was significantly lower in HF group. 2.After rosiglitazone intervention, the levels of fasting blood glucose,triglyceride,cholesterol,FFAs,fasting insulin and relative visceral fat mass were lower in RSG group and glucose infusion rate was higher than those in HF group. 3. Compared with NC group, the mRNA and protein expression of ATGL and HSL were decreased in HF group (P<0.05). After rosiglitazone intervention, there were no significance in ATGL and HSL expression between HF and RGS group despite mild increases in those indications (P>0.05). 4. Fasting insulin and FFAs were negatively correlated with ATGL protein expression (r=-0.379 and r=-0.454,P<0.01), and glucose infusion rate was in positive correlation with ATGL protein expression (r=0.354, P<0.05). Fasting insulin and FFAs were negatively correlated with HSL protein expression (r=-0.356 and r=-0.324,P<0.01), and glucose infusion rate was positively correlated with HSL protein expression (r=0.277, P<0.05). 5. ATGL protein expression and HSL protein expression were in positive correlation (r=0.603, P<0.001).Summary: 1.High-fat diets induced insulin resistance of rats in the presentations of increased fasting blood glucose,triglyceride,total cholesterol,FFAs and fasting insulin and decreased glucose infusion rate. Oral glucose tolerance test demonstrated impaired function of dealing with glucose load by high-fat diets. 2.ATGL and HSL expression were decreased in adipose tissue of high-fat feeding rats, suggesting there was a disordered fat mobilization and may attenuate FFAs release at the early stage of insulin resistance. 3. Rosiglitazone improved the glucose and lipid metabolism of high-fat feeding rats and altered the expression of ATGL and HSL in adipose tissue. Part Two: Study on the expression of ATGL and HSL in adipose tissue of insulin-resistant peopleObjective : To investigate the mRNA and protein expression of ATGL and HSL in subcutaneous and visceral fat of insulin-resistant people, and to explore the relationship between disordered fat mobilization, fat-depot specificity and insulin resistance.Methods: All subjects were collected from inpatients who was going to have selective cholecystectomy and signed the protocol. Based on the HOMA-IR (homeostasis model assessment-insulin resistance), all cases were divided into insulin sensitive group (ISG) and insulin resistant group (IRG). Anthropometric parameters and biochemical indicators were determined before operation. Subcutaneous and visceral adipose tissue was taken on the operation day with liquid nitrogen and stored in -70℃refrigerator. RT-PCR was used to examine the mRNA expression of ATGL and HSL; while western blot analysis was used to evaluate protein expression. Results: 1. Independent of gender and age, body mass index, waist-to-hip ratio, triglyceride, fasting insulin and free fatty acids (FFA) were higher in IRG than those in ISG. 2. Compared with IS group, ATGL and HSL protein and mRNA expression were decreased in IR group in both visceral and subcutaneous adipose tissue. 3. Compared with IS group, HSL protein expression and mRNA expression were decreased in visceral fat of IR group (P<0.05); there was no significance in HSL protein expression and mRNA expression in subcutaneous fat between the two groups. 4. Fasting insulin, HOMA-IR and FFA were in negative correlation with ATGL protein expression (r=-0.235, r=-0.356 and r=-0.214,P<0.05); while fasting insulin, FFA and waist-to-hip ratio were negatively correlated with HSL protein expression (r=-0.185, r=-0.246 and r=-0.145,P<0.05). 5. ATGL and HSL protein expression were in high positive correlation in both visceral and subcutaneous adipose tissue (r=0.708 and r=0.632,P<0.001).Summary: 1. The expression of ATGL and HSL was decreased in visceral adipose tissue of insulin-resistant people;ATGL expression was decreased in subcutaneous fat of insulin-resistant people. 2. ATGL protein expression and HSL protein expression were in high positive correlation, suggesting they belonged to a common regulatory pathway. 3. There was difference of ATGL and HSL expression between visceral and subcutaneous adipose tissue, suggesting that fat-depot specificity may play roles in the development of insulin resistance.Conclusion:1. High-fat diet induced insulin resistance of rats. 2. ATGL and HSL expression were decreased in adipose tissue in both high-fat feeding rats and insulin-resistant people. 3. ATGL protein expression and HSL protein expression were in high positive correlation, suggesting they belonged to a common regulatory pathway. 4. Expressions of ATGL and HSL in subcutaneous adipose tissue and visceral fat were different, suggesting fat-depot specificity may play roles in the development of insulin resistance. 5. Rosiglitazone improved the glucose and lipid metabolism of high-fat feeding rats and influenced the expression of ATGL and HSL in adipose tissue.